Small Ubiquitin-like Modifier (SUMO) proteins are a family of proteins which are structurally similar to ubiquitin. In lower eukaryotes a single SUMO gene is expressed (Smt3 in Saccharomyces cerevisiae), whereas in vertebrates three paralogs designated SUMO1, SUMO2 and SUMO3 are ubiquitously expressed in all tissues. The human genome also encodes a forth gene for SUMO4 that appears to be uniquely expressed in the spleen, lymph nodes and kidney (Guo, D. et al. A functional variant of SUMO4, a new I kappa B alpha modifier, is associated with type 1 diabetes. Nat Genet 36, 837-841 (2004)), though its in vivo maturation into a conjugation-competent form still remains unclear (Owerbach, D., McKay, E. M., Yeh, E. T., Gabbay, K. H. & Bohren, K. M. A proline-90 residue unique to SUMO-4 prevents maturation and sumoylation. Biochem Biophys Res Commun 337, 517-520 (2005)). Protein SUMOylation is the post-translational covalent but reversible conjugation of SUMO (SUMO-1, 2 and 3 isoforms in mammalian cells) to protein substrates. This covalent modification is obtained by the formation of an isopeptide bond between the ε-amino group of a lysine residue from the protein substrate and the C-terminus COOH group of the SUMO isoform. This modification is structurally similar to ubiquitin although it shares less than 20% amino acid sequence homology.
Protein SUMOylation is an essential cellular process conserved from yeast to mammals. It is involved in different processes including the regulation of intracellular trafficking, cell cycle, DNA repair and replication, RNA metabolism, cell signaling and stress responses (Bossis, G., and Melchior, F. (2006). SUMO: regulating the regulator. Cell division 1, 13; Hay, R. T. (2005). SUMO: a history of modification. Molecular cell 18, 1-12).
Protein SUMOylation imparts significant structural and conformational changes on the substrate protein by masking and or by conferring additional scaffolding surfaces for protein interactions.
At present, several hundred protein substrates are known to be SUMOylated. These protein targets include regulators of gene expression (e.g. transcription factors, co-activators or repressors) as well as oncogenes and tumor suppressor genes, such as promyelocytic leukaemia (PML), Mdm2, c-Myb, c-Jun, and p53 whose misregulation leads to tumorigenesis and metastasis (Kim, K. I., and Baek, S. H. (2006). SUMOylation code in cancer development and metastasis. Molecules and cells 22, 247-253).
Protein SUMOylation is a highly dynamic modification regulated by a complex network of SUMO-activating enzymes (SAE1/SAE2), conjugating enzymes (Ubc9) and SUMO-E3 ligases (PIAS1, PIAS3, PIASxα, PIASxβ, PIASy, RanBP2 and Pc2) for the transfer of SUMO isoforms to specific protein substrates (Kim, K. I., and Baek, S. H. (2006). SUMOylation code in cancer development and metastasis. Molecules and cells 22, 247-253; Guo, B., Yang, S. H., Witty, J., and Sharrocks, A. D. (2007). Signalling pathways and the regulation of SUMO modification. Biochemical Society transactions 35, 1414-1418). The dynamic changes in protein SUMOylation in response to different cell stimuli is counter-balanced by SUMO-specific proteases (SUSP's or SENPs) which cleave this modification on specific SUMO substrates (see FIG. 1).
Currently, the extent and biological significance of protein SUMOylation in cell regulation and cancer development, remains poorly understood. No efficient methods exist for the comprehensive quantitation and analysis of this modification from cell extracts. The relatively low stoichiometry of protein SUMOylation is a significant analytical challenge for its identification and quantitation in intact cells. Recent reports have described the successful identification of SUMO protein candidates by transfecting His6-SUMO1 and His6-SUMO-2, and quantifying their proportions using mass spectrometry (MS) and metabolic labelling in cell cultures (Vertegaal, A. C., Andersen, J. S., Ogg, S. C., Hay, R. T., Mann, M., and Lamond, A. I. (2006). Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics. Mol Cell Proteomics 5, 2298-2310).
However, the identification of SUMOylation sites by MS remains challenging due to their low occurrence and the presence of long SUMO C-termini polypeptides which lack Arg/Lys. This complicates the MS/MS assignment of the corresponding tryptic peptides (Pedrioli, P. G., Raught, B., Zhang, X. D., Rogers, R., Aitchison, J., Matunis, M., and Aebersold, R. (2006). Automated identification of SUMOylation sites using mass spectrometry and SUMmOn pattern recognition software. Nature methods 3, 533-539).
Furthermore, the lack of efficient tools and methods to identify protein SUMOylation also complicates the identification of enzymes responsible for this modification and of substrates upon which they act. Thus, there is a need for new methods to identify protein SUMOylation sites.